Conocephalum salebrosum, Szweyk., Buczk. & Odrzyk

Zhang, Chi, Chen, Xinlu, Crandall-Stotler, Barbara, Qian, Ping, Köllner, Tobias G., Guo, Hong & Chen, Feng, 2019, Biosynthesis of methyl (E) - cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase, Phytochemistry 164, pp. 50-59 : 51-53

2.1. Production of axenic culture of C. salebrosum and chemical profiling

The sample collected from Illinois (IL) morphologically matched C. salebrosum , a segregate species of C. conicum ( Szweykowski et al., 2005) . This population is the chemotype with methyl (E)-cinnamate as the dominant compound described by Wood et al. (1996), and was therefore chosen as the model chemotype for most of the experiments in this study. Live plants were collected in the field and grown in axenic culture. Chemical profiling analysis was performed using gas chromatography–mass spectrometry (GC-MS) to determine the volatile components in the axenically grown gametophytes of this species of the C. conicum complex. From organic extraction, a total of 10 volatiles were detected ( Fig. 1A ), of which methyl (E)-cinnamate was the dominant component, accounting for 50.5% of the total volatiles. The monoterpene sabinene was the second most abundant with a concentration of 20% of that of methyl (E)-cinnamate. The thallus of C. salebrosum , when growing in the wild, possesses a characteristic aromatic odor. To determine whether C. salebrosum grown axenically on culture medium emits volatiles, we performed headspace collection of explants of C. salebrosum , which was analyzed by GC-MS. Three dominant compounds were detected ( Fig. 1B ). All of them were also detected by organic extraction. Similar to organic extraction, methyl (E)-cinnamate was the most abundant compound in the volatile bouquet of C. salebrosum ( Fig. 1B ).

2.2. Isolation of SABATH genes from C. salebrosum and gene expression analysis

We hypothesized that in the C. salebrosum methyl (E)-cinnamate is synthesized by the action of SABATH methyltransferase. To identify SABATH genes, we analyzed the transcriptome of C. conicum prepared from vegetative thalli (https://sites.google.com/a/ualberta.ca/onekp), which was produced by the 1 KP consortium ( Matasci et al., 2014). Via a HMMR search ( Finn et al., 2011) using Methyltransf_7 Pfam profile ( Finn et al., 2015) as query, a total of nine unigenes encoding SABATH proteins were identified. Six of them appear to be in full-length when compared to other SABATH proteins and their corresponding orthologs in C. salebrosum were designated CsSABATH1-6 ( Table S1 ). Using RT-PCR, full-length cDNAs for all six CsSABATH genes were cloned from the thallus of the axenically grown C. salebrosum . The lengths of six proteins range from 383 to 443 amino acids ( Fig. 2 ). Sequence similarities among CsSABATHs range from 37% to 52%. Their similarities to ObCCMT1, one known CAMT from basil, are between 36% and 48%. The amino acid residues for binding the methyl donor SAM among CsSABATHs and ObCCMT1 are identical. The residues that interact with the carboxyl moiety of the substrate are also conserved. However, the residues that interact with the aromatic moiety are significantly different ( Fig. 2 ).

With six SABATH genes cloned from C. salebrosum , next we compared their expression levels using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. At 25 cycles of

PCR reactions, transcripts for five of the six genes could be detected from thallus tissue, the exception being CsSABATH1 ( Fig. 3 ). CsSABATH4 showed the highest level of expression. While CsSABATH2 and CsSABATH6 showed also higher expression levels. CsSABATH3 and CsSABATH5 were found to be expressed at relatively low levels ( Fig. 3 ).

Figures

Fig. 1. Chemical analysis of C. salebrosum. (A) GC chromatogram of the organic extraction of C. salebrosum. 1*, sabinene; 2, 1-octanol; 3, 1-octenyl acetate; 4*, methyl (E)-cinnamate; 5, unidentified sesquiterpene hydrocarbon; 6, 10-epi- cubebol; 7, 1-epi-cubenol; 8, 1-octadecyne; 9, (E)-2-tetradecen-1-ol; 10, phytol. (B) GC chromatogram of headspace collection of C. salebrosum. 1*, sabinene; 4*, methyl (E)-cinnamate; 5, unidentified sesquiterpene hydrocarbon. “*” indicates compounds that were verified by authentic standards.

Fig. 2. Sequence alignment of CcSABATHs with ObCCMT1 and CbSAMT. Conserved residues are in shade with the more conserved the darker. Residues indicated with “&” are SAM/SAH-binding residues. Residues indicated with “*” are residues that interact with the carboxyl moiety of substrate. Residues indicated with “#” interact with the aromatic moiety of substrate and are important for substrate selectivity. CcSABATHs, C. conicum sensu latu SABATHs; ObCCMT1, Ocimum basilicum cinnamate/p-coumarate carboxyl methyltransferase 1; CbSAMT, Clarkia breweri salicylic acid methyltransferase.

Fig. 3. Expression of six CsSABATHs genes in sterile thalli measured by semiquantitative RT-PCR. Same amount of cDNA was used in each reaction. This experiment was repeated three times with similar results. “M” indicates molecular marker.